Project Summary Most fibroblast growth factor (FGF) proteins are secreted proteins that act as ligands for cognate receptors (FGFRs) expressed on other cells. However, we hypothesize that FGFs have additional functions. The experiments proposed here will investigate a noncanonical role for FGFs in supporting a response in the ligand- expressing cell, to determine if FGFs also ?reverse signal?. Reverse signaling has been linked to several other signaling pathways including ephrin, semaphorin, and TNFa, but not previously demonstrated for FGFs. We study FGF signaling in Drosophila, a model system that we contend is poised to provide novel insights into FGF signaling. The Drosophila FGF signaling system of Drosophila is less complex, with only 3 receptor-ligand combinations functional compared to over 100 combinations in vertebrates. Nevertheless, even in the simpler Drosophila system, it has remained unclear whether individual ligands have different activities. To provide insight, preliminary structure-function analyses of two Drosophila FGF proteins Pyramus (Pyr) and Thisbe (Ths) were conducted. We uncovered differences in protein topology that suggest these proteins have distinct activities including, specifically, that one ligand functions to ?signal in reverse?. Drosophila is an excellent model system to analyze whether reverse signaling by FGFs exists and also to determine whether it has a functional role in support of proper embryonic development. To provide insight, our proposed study has three specific aims. In Aim 1, a structure-function analysis of FGF proteins will be undertaken, in which we will investigate localization and function of FGF protein domains within embryos to provide insight into distinct activities of the N- and C- termini in the extracellular and intracellular spaces, respectively. In Aim 2, experiments are proposed to identify the mechanism of action used by FGFs to reverse signaling in vivo. We will investigate FGF-interacting proteins using mass spectrometry to provide insight into function of the Pyr intracellular C-terminal domain. In Aim 3, experiments are proposed to test the specific hypothesis that Heartless FGFR and/or decoy receptor FGFRL1 act as ?ligands? for Pyr to support its reverse signaling. The demonstration that a Drosophila FGF signals in reverse would suggest that some vertebrate FGFs also exhibit this activity. A better understanding of the full capacity of FGF protein function has the potential to provide insight towards gene therapies for congenital diseases that relate to aberrant FGF signaling, as drug candidates that target particular FGF-associated activities will likely have fewer side effects. As FGF signaling plays an important and pervasive role during development of all metazoan animals including humans, insights gained into the regulation of FGF activity from this proposed work are likely to have far-reaching impact.